Circadian control of the daily rhythm of adult emergence by regulation of the timing of ecdysone action in Drosophila melanogaster



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Universidad de Valparaíso



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Instituto de Neurociencia




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In Drosophila melanogaster, the circadian clock imposes a daily rhythm to the pattern of adult emergence (eclosion) by a process that has been described as “gating”. My research is aimed at identifying the mechanism by which the daily “gating” of the time of emergence occurs. In this insect, the circadian clock sets the time of emergence through the coupling between the central clock located in the brain and a peripheral clock contained in the Prothoracic Gland (PG), an endocrine gland whose only known function is the production of the molting hormone, ecdysone (E). The levels of E increase to cause the larval molts and metamorphosis, and then drop to signal the end of each molt. Previous work in the insect, Rhodnius prolixus, suggests that ecdysone is involved in the gating of eclosion because injections of increasing doses of E delay the time of eclosion to later times in a continuous fashion, suggesting that the titer of E itself is central to the gating mechanism. Moreover, in Rhodnius, molting is accompanied by circadian oscillations in ecdysone titers, produced as a consequence of its rhythmic release. Nevertheless, this rhythm of E does not seem to be a general feature among insects. In Drosophila, in particular, no circadian oscillations in E have been detected during metamorphosis. However, whether the clock regulates ecdysone signaling through a different mechanism, remains to be explored. Thus, the main goal of my Doctoral thesis is to use Drosophila to explore the role of E in the circadian gating of eclosion by asking whether there are elements of the E pathway that are under circadian control and what are the molecular mechanisms used by the clock to impose a daily rhythmicity to adult emergence. To address this question, in Chapter 2 I first asked whether the clock regulates the time when flies commit to end metamorphosis and whether the clock can set the time of adult emergence by regulating ecdysone levels. Since my results support a mechanism where the clock acts downstream of E, I also disrupted E signaling in the PG via its receptor, EcR, and evaluated the consequences of these manipulations on the rhythm of eclosion and locomotor activity. Next, in Chapter 3 I evaluated the role of EcR in the circadian regulation of the rhythm of emergence. For this purpose, I used immunohistochemistry and fluorescent in situ hybridization to determine whether the clock regulates the expression of EcR during the course of the day. Moreover, I analyzed whether the clock acts by synchronizing the cellular size within the PG (as a proxy for cellular activity) and, in addition, I determined which are the possible molecular mechanisms used by the clock to regulate EcR action. Finally, in Chapter 4 I asked whether there are other elements of the E transduction pathway that may be under circadian control. For this I assessed the effects on the rhythm of eclosion of downregulating in the PG candidate genes involved in transducing 20E actions. Results: I found that the time when flies initiate the final steps of metamorphosis is correlated with the time of emergence, which suggests that the circadian clock gates emergence by controlling the time when the animal commits to complete metamorphosis, and not by simply preventing the emergence of animals that completed metamorphosis before the gate opens (work done in collaboration with Brandon Mark, Guadalupe Cascallares, and Felipe Conejera). Moreover, I found that injecting increasing doses of E prior to eclosion produced a dose-dependent delay in the time of adult emergence but did not disrupt its circadian rhythmicity, which suggests that the clock acts downstream of E. Consistent with this hypothesis, I found that interfering with E signaling in the PG renders arrhythmic the pattern of emergence. In addition, my findings reveal that the circadian clock controls EcR function by regulating its intracellular location as well as its level of expression. Finally, I found that downregulating genes directly induced by ecdysone also eliminated the rhythm of eclosion. Conclusions: My results suggest that the circadian clock imposes a daily rhythm to the pattern of emergence by regulating the process of E signal transduction in the PG. The study of the pathways linking development and the circadian system will help elucidate how the clock regulates the timing of emergence, in addition to helping in the understanding of how daily steroid hormone rhythms are generated in animals.


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• CONYCIT Doctoral Fellowship N°21191590 • FONDECYT N°1180403 • Centro Interdisciplinario de Neurociencia de Valparaíso (CINV) Millennium Institute grant P09-022-F • FIB-UV Doctoral Fellowship from Universidad de Valparaíso

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